A mutation leading to a stop codon in the FVIII gene is the cause of severe canine Hemophilia A

Abstract number: P0672

Wilhelm^* C., Czwalinna^* A., Hoffmann^* M., Mischke† R., Ganser^* A., Von Depka^* M.

^*Hannover Medical School, Germany; †Hannover Veterinarian School, Germany

Introduction  

Factor VIII (FVIII) deficiency is the reason for Hemophilia A. Severe Hemophilia A has been reported in dogs and only a few mutations in the canine FVIII gene are known which may cause this disease.

Materials and methods  

We analyzed blood samples of a German shepherd dog with FVIII plasma levels <1% of normal canine levels. The coding region of the canine FVIII gene comprises a 7032-bp nucleotide sequence. First, we tried to isolate the canine FVIII mRNA from liver to amplify the canine cDNA by RT-PCR. However, it was impossible to amplify a product. We therefore isolated the genomic DNA from EDTA blood samples and sequenced the exons of the FVIII gene. The investigated regions of the FVIII gene was amplified by PCR, and DNA sequencing was performed on both strands using canine FVIII primers for the amplified products by use of the dye-deoxy terminator method and an automated DNA sequencer system (Applied Biosystems).

Results  

We sequenced exon 1 to exon 24 of the canine FVIII gene and found only a newly identified single mutation TGG ® TAG at nucleotide position 98 in exon 1 resulting in a STOP codon. No further mutation could be detected in the genomic DNA sequence of the coding region. The Trp33Stop mutation leads to a short protein truncated after 33 amino acids containing no functional FVIII domains.

Conclusion  

We suggest that this mutation is responsible for the severe FVIII deficiency in the dog. The early introduction of a STOP codon at position 33 explains the fact that we were unable to amplify cDNA products by RT-PCR due to the lack of canine FVIII mRNA.